Tumor necrosis factor xcex1 (TNFxcex1) is a cytokine produced by numerous cell types, including monocytes and macrophages, that was originally identified based on its capacity to induce the necrosis of certain mouse tumors (see e.g., Old, L. (1985) Science 230:630-632). Subsequently, a factor termed cachectin, associated with cachexia, was shown to be the same molecule as TNFxcex1. TNFxcex1 has been implicated in mediating shock (see e.g., Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518; Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655). Furthermore, TNFxcex1 has been implicated in the pathophysiology of a variety of other human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection and graft-versus-host disease (see e.g., Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).
Because of the harmful role of human TNFxcex1 (hTNFxcex1) in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract hTNFxcex1 activity. In particular, antibodies that bind to, and neutralize, hTNFxcex1 have been sought as a means to inhibit hTNFxcex1 activity. Some of the earliest of such antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with hTNFxcex1 (see e.g., Hahn T; et al., (1985) Proc Natl Acad Sci USA 82: 3814-3818; Liang, C-M., et al. (1986) Biochem. Biophys. Res. Commun. 137:847-854; Hirai, M., et al. (1987) J. Immunol. Methods 96:57-62; Fendly, B. M., et al. (1987) Hybridoma 6:359-370; Mxc3x6ller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; European Patent Publication No. 186 833 B1 by Wallach, D.; European Patent Application Publication No. 218 868 A1 by Old et al.; European Patent Publication No. 260 610 B1 by Moeller, A., et al.). While these mouse anti-hTNFxcex1 antibodies often displayed high affinity for hTNFxcex1 (e.g., Kdxe2x89xa610xe2x88x929M) and were able to neutralize hTNFxcex1 activity, their use in vivo may be limited by problems associated with administration of mouse antibodies to humans, such as short serum half life, an inability to trigger certain human effector functions and elicitation of an unwanted immune response against the mouse antibody in a human (the xe2x80x9chuman anti-mouse antibodyxe2x80x9d (HAMA) reaction).
In an attempt to overcome the problems associated with use of fully-murine antibodies in humans, murine anti-hTNFxcex1 antibodies have been genetically engineered to be more xe2x80x9chuman-like.xe2x80x9d For example, chimeric antibodies, in which the variable regions of the antibody chains are murine-derived and the constant regions of the antibody chains are human-derived, have been prepared (Knight, D. M, et al. (1993) Mol. Immunol. 30:1443-1453; PCT Publication No. WO 92/16553 by Daddona, P. E., et al.). Additionally, humanized antibodies, in which the hypervariable domains of the antibody variable regions are murine-derived but the remainder of the variable regions and the antibody constant regions are human-derived, have also been prepared (PCT Publication No. WO 92/11383 by Adair, J. R., et al.). However, because these chimeric and humanized antibodies still retain some murine sequences, they still may elicit an unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction, especially when administered for prolonged periods, e.g., for chronic indications, such as rheumatoid arthritis (see e.g., Elliott, M. J., et al. (1994) Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet 344:1105-1110).
A preferred hTNFxcex1 inhibitory agent to murine mAbs or derivatives thereof (e.g., chimeric or humanized antibodies) would be an entirely human anti-hTNFxcex1 antibody, since such an agent should not elicit the HAMA reaction, even if used for prolonged periods. Human monoclonal autoantibodies against hTNFxcex1 have been prepared using human hybridoma techniques (Boyle, P., et al. (1993) Cell. Immunol. 152:556-568; Boyle, P., et al. (1993) Cell. Immunol. 152:569-581; European Patent Application Publication No. 614 984 A2 by Boyle, et al.). However, these hybridoma-derived monoclonal autoantibodies were reported to have an affinity for hTNFxcex1 that was too low to calculate by conventional methods, were unable to bind soluble hTNFxcex1 and were unable to neutralize hTNFxcex1-induced cytotoxicity (see Boyle, et al.; supra). Moreover, the success of the human hybridoma technique depends upon the natural presence in human peripheral blood of lymphocytes producing autoantibodies specific for hTNFxcex1. Certain studies have detected serum autoantibodies against hTNFxcex1 in human subjects (Fomsgaard, A., et al. (1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog. Leukocyte Biol. 10B:447-452), whereas others have not (Leusch, H-G., et al. (1991) J. Immunol. Methods 139:145-147).
Alternative to naturally-occurring human anti-hTNFxcex1 antibodies would be a recombinant hTNFxcex1 antibody. Recombinant human antibodies that bind hTNFxcex1 with relatively low affinity (i.e., Kdxcx9c10xe2x88x927M) and a fast off rate (i.e., Koffxcx9c10xe2x88x922 secxe2x88x921) have been described (Griffiths, A. D., et al. (1993) EMBO J. 12:725-734). However, because of their relatively fast dissociation kinetics, these antibodies may not be suitable for therapeutic use. Additionally, a recombinant human anti-hTNFxcex1 has been described that does not neutralize hTNFxcex1 activity, but rather enhances binding of hTNFxcex1 to the surface of cells and enhances internalization of hTNFxcex1 (Lidbury, A., et al. (1994) Biotechnol. Ther. 5:27-45; PCT Publication No. WO 92/03145 by Aston, R. et al.)
Accordingly, human antibodies, such as recombinant human antibodies, that bind soluble hTNFxcex1 with high affinity and slow dissociation kinetics and that have the capacity to neutralize hTNFxcex1 activity, including hTNFxcex1-induced cytotoxicity (in vitro and in vivo) and hTNFxcex1-induced cell activation, are still needed.
This invention provides human antibodies, preferably recombinant human antibodies, that specifically bind to human TNFxcex1. The antibodies of the invention are characterized by binding to hTNFxcex1 with high affinity and slow dissociation kinetics and by neutralizing hTNFxcex1 activity, including hTNFxcex1-induced cytotoxicity (in vitro and in vivo) and hTNFxcex1-induced cellular activation. The antibodies can be full-length (e.g., an IgG1 or IgG4 antibody) or can comprise only an antigen-binding portion (e.g., a Fab, F(abxe2x80x2)2 or scFv fragment). The most preferred recombinant antibody of the invention, termed D2E7, has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4. Preferably, the D2E7 antibody has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2.
In one embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNFxcex1 with a Kd of 1xc3x9710xe2x88x928 M or less and a Koff rate constant of 1xc3x9710xe2x88x923 sxe2x88x921 or less, both determined by surface plasmon resonance, and neutralizes human TNFxcex1 cytotoxicity in a standard in vitro L929 assay with an IC50 of 1xc3x9710xe2x88x927 M or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, dissociates from human TNFxcex1 with a Koff of 5xc3x9710xe2x88x924 sxe2x88x921 or less, or even more preferably, with a Koff of 1xc3x9710xe2x88x924 sxe2x88x921 or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, neutralizes human TNFxcex1 cytotoxicity in a standard in vitro L929 assay with an IC50 of 1xc3x9710xe2x88x928 M or less, even more preferably with an IC50 of 1xc3x9710xe2x88x929 M or less and still more preferably with an IC50 of 1xc3x9710xe2x88x9210 M or less.
In another embodiment, the invention provides a human antibody, or antigen-binding portion thereof, with the following characteristics:
a) dissociates from human TNFxcex1 with a Koff of 1xc3x9710xe2x88x923 sxe2x88x921l or less, as determined by surface plasmon resonance;
b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;
c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
More preferably, the antibody, or antigen-binding portion thereof, dissociates from human TNFxcex1 with a Koff of 5xc3x9710xe2x88x924 sxe2x88x921 or less. Still more preferably, the antibody, or antigen-binding portion thereof, dissociates from human TNFxcex1 with a Koff of 1xc3x9710xe2x88x924 sxe2x88x921 or less.
In yet another embodiment, the invention provides a human antibody, or an antigen-binding portion thereof, with an LCVR having CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8, and with an HCVR having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. More preferably, the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 and the HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6. Still more preferably, the LCVR further has CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 and the HCVR has a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8.
In still another embodiment, the invention provides an isolated human antibody, or an antigen binding portion thereof, with an LCVR comprising the amino acid sequence of SEQ ID NO: 1 and an HCVR comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the antibody has an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In yet other embodiments, the antibody is a Fab fragment, an F(abxe2x80x2)2 fragment or a single chain Fv fragment.
In still other embodiments, the invention provides antibodies, or antigen-binding portions thereof, with an LCVR having CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or with an HCVR having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.
In yet another embodiment, the invention provides an isolated human antibody, or antigen-binding portion thereof, that neutralizes the activity of human TNFxcex1, chimpanzee TNFxcex1 and at least one additional primate TNFxcex1 selected from the group consisting of baboon TNFxcex1, marmoset TNFxcex1, cynomolgus TNFxcex1 and rhesus TNFxcex1. In one subembodiment, the isolated human antibody, or antigen-binding portion thereof, also neutralizes the activity of mouse TNFxcex1. In another subembodiment, the isolated human antibody, or antigen-binding portion thereof, also neutralizes the activity of pig TNFxcex1.
Another aspect of the invention pertains to nucleic acid molecules encoding the antibodies, or antigen-binding portions, of the invention. A preferred nucleic acid of the invention, encoding a D2E7 LCVR, has the nucleotide sequence shown in FIG. 7 and SEQ ID NO: 36. Another preferred nucleic acid of the invention, encoding a D2E7 HCVR, has the nucleotide sequence shown in FIG. 8 and SEQ ID NO: 37. Recombinant expression vectors carrying the antibody-encoding nucleic acids of the invention, and host cells into which such vectors have been introduced, are also encompassed by the invention, as are methods of making the antibodies of the invention by culturing the host cells of the invention.
Yet another aspect of the invention pertains to methods for inhibiting human TNFxcex1 activity using an antibody, or antigen-binding portion thereof, of the invention. In one embodiment, the method comprises contacting human TNFxcex1 with the antibody of the invention, or antigen-binding portion thereof, such that human TNFxcex1 activity is inhibited. In another embodiment, the method comprises administering an antibody of the invention, or antigen-binding portion thereof, to a human subject suffering from a disorder in which TNFxcex1 activity is detrimental such that human TNFxcex1 activity in the human subject is inhibited. The disorder can be, for example, sepsis, an autoimmune disease (e.g., rheumatoid arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome), an infectious disease, a malignancy, transplant rejection or graft-versus-host disease, a pulmonary disorder, a bone disorder, an intestinal disorder or a cardiac disorder.